DESCRIPTION: Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are an important therapeutic class of drugs that are widely used in antiretroviral therapy strategies to treat and prevent HIV-1 infection. They bind to a hydrophobic pocket in HIV-1 RT, termed the NNRTI-binding pocket (BP), which is located ~ 10 away from the polymerase active site of the enzyme. Our current understanding of how NNRTIs inhibit HIV-1 reverse transcription, and how mutations in the NNRTI-BP confer inhibitor resistance, has been largely inferred from crystal structures of HIV-1 RT in complex with NNRTIs. We have only limited knowledge in regard to how NNRTIs affect the catalytically relevant RT-template/primer (T/P) binary and RT-T/P-dNTP ternary complexes. Importantly, single-molecule Fster resonance energy transfer studies revealed that NNRTI-binding to RT can impact the binding orientation and sliding dynamics of RT on the T/P substrate. Crystallography cannot provide insight into the dynamic interactions between biomolecules. Indeed, there are only 2 crystal structures available of an NNRTI-bound RT-T/P binary complex (and none for the ternary complex) - and in one of these structures RT is cross-linked to the T/P substrate. As such, there is a critical knowledge gap in regard to how NNRTI-binding to wild-type and mutant RT impacts: (i) the dynamic inter-molecular interactions between the enzyme and its substrates; and (ii) the intra-molecular protein conformational changes in RT-T/P and RT-T/P- dNTP complexes. The primary goal of this application is to address these knowledge gaps using state-of-the- art single-molecule and ensemble biophysical approaches (developed in our laboratories) that can quantitatively assess the dynamic inter-molecular interactions between HIV-1 RT and its substrates and the intra-molecular conformational changes in RT. We anticipate that the data derived from these studies will provide unprecedented mechanistic insight into the mode of action of NNRTIs and the mechanisms associated with NNRTI resistance. Collectively these studies may significantly impact future drug discovery efforts.